CN105699667B - Bacterial magnetic particles erythrocyte membrane composite particles and its clinical practice - Google Patents

Bacterial magnetic particles erythrocyte membrane composite particles and its clinical practice Download PDF

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CN105699667B
CN105699667B CN201610053399.9A CN201610053399A CN105699667B CN 105699667 B CN105699667 B CN 105699667B CN 201610053399 A CN201610053399 A CN 201610053399A CN 105699667 B CN105699667 B CN 105699667B
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erythrocyte membrane
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CN105699667A (en
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张金菊
王红光
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BEIJING GUO KE RONG ZHI BIOTECHNOLOGY Co.,Ltd.
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Beijing Zhongke Yuanrong Biological Technology Development Co Ltd
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Abstract

It is by by bacterial magnetic particles surface NH to preserve with application in blood group antibody detection, the composite particles the invention discloses a kind of bacterial magnetic particles erythrocyte membrane composite particles and its in erythrocyte membrane antigen2Group makes its activation by processing, is incubated jointly with erythrocyte membrane, bacterial magnetic particles surface is combined erythrocyte membrane；The bacterial magnetic particles erythrocyte membrane composite particles are used to preserve human erythrocyte membrane blood group antigens for a long time and for the detection of blood group antibody, a kind of quick, easy test method can be provided for clinic；Advantage of this approach is that bacterial magnetic particles are more homogeneous compared with magnetic particle performance, the size of chemical synthesis, source is unrestricted, biocompatibility is more preferable, the epitope activity of Surface of Erythrocytes blood group antigens can be ensured with erythrocyte membrane strong bonded and to greatest extent, its bacterial magnetic particles erythrocyte membrane composite particles prepared, it is particularly conducive to the preservation of clinical blood group antigen and the detection of blood group antibody.

Description

Bacterial magnetic particles erythrocyte membrane composite particles and its clinical practice
Technical field
The present invention relates to field of medical examination, more particularly to bacterial magnetic particles erythrocyte membrane composite particles and its red thin Application in membrane antigenses preservation and blood group antibody detection.
Background technology
Erythrocyte blood type is one of human inheritance's multiformity mark, since early 20th century finds abo blood group, it has been found that Individual human erythrocyte's blood group antigens more than 500, more than 200 is had confirmed that at present.Wherein, include with clinical blood group system the closest 29 systems such as ABO, Rh, MNS.Clinical blood group serology detection mainly has：Blood grouping, antibody screening and identification, intersection are matched somebody with somebody Blood.At present, red blood cell used in blood group serology detection is fresh cells.But fresh red blood cell storage life is short, only 3 Month, and in use, it is easily influenced by environmental conditions, it is difficult to meet routine clinical detection application.
Magnetotactic bacteria (Magnetotactic bacteria, MTB) is that one kind can arrange or orient along magnetic line of force direction Motion, the gramnegative bacterium for synthesizing the bacterial magnetic particles surrounded by biomembrane in the cell, it is generally distributed in nature, But because it is very harsh to growth conditions, nutritional requirement so that its pure culture and the research of characteristic are extremely difficult.In 80 years The research of generation and the initial stage nineties to magnetotactic bacteria once state at a low ebb.In recent years, with Protocols in Molecular Biology etc. Develop rapidly, the research to the morphology, biological characteristics, condition of culture of magnetotactic bacteria etc. also develops rapidly.
Magnetotactic bacteria is that substantial amounts of iron is absorbed from environment, forms a kind of special organelle, i.e. bacterial magnetic particles, in it Contain the ferroso-ferric oxide (Fe of a single magnetic domain in portion3O4) or ferriferrous sulfide (Fe3S4) crystal, have lipid and egg outside it The elementary membrane coating formed in vain.Different bacterial strains can synthesize bacterial magnetic particles of different shapes, have species specificity.Bacterial magnetic Particle mainly has truncated octahedra, prism-shaped, bullet shaped, arrow shaped, dentation etc., 30~120nm of diameter.In the cell, bacterium It is arranged in one or several chain the usual high-sequential of magnetic particle, forms one or more " small magnetic needles ", so that thalline can be with Perceive external magnetic field；The forming process of bacterial magnetic particles belongs to biomineralization (biomineralization), and its whole process is more Carried out in 50nm or so vesicle, by strict bioelectric detecting, mild condition, product is single magnetic domain crystal, uniform particle diameter, Stable crystal formation, magnetic is most strong in same type of material, is qualified " nanometer factory (Nanofactory) ".Meanwhile bacterial magnetic Particle has elementary membrane coating, and compared with exposed magnetic material, it has superparamagnetism, is easily dispersed, and there is no method artificial at present Simulation.In addition, bacterial magnetic particles are uniform in size, surface area is big, and there is biomembrane coating outside, be not easy to assemble between particle, have There are good dispersiveness and biocompatibility.
Bacterial magnetic particles are a kind of natural magnetism nano meter biomaterials, are had due to its excellent performance in many fields Huge potential using value, such as the disclosed anti-coated bacterial magnetics of Bt insecticidal proteins polyclonal antibody of CN102419370 Particle can be used for Bt insecticidal proteins in detection mouse tissue；CN101077418 is disclosed to carry adriamycin bacteria nano bacterial magnetic Grain can be used for suppressing propagation of cancer cell etc.；But prior art is not disclosed also and is used in combination using bacterial magnetic particles and erythrocyte membrane In the relevant report of blood group antibody detection.
The content of the invention
In order to solve the above-mentioned technical problem, the present invention provides a kind of bacterial magnetic particles erythrocyte membrane composite particles and its red Application in membrane antigen preservation and blood group antibody detection, the detection method is easy, efficient, as a result accurately, reliably.
Concrete technical scheme of the present invention is as follows：
One aspect of the present invention provides a kind of bacterial magnetic particles erythrocyte membrane composite particles, and the wherein particle is by surface NH2 Group activated by coupling agent after bacterial magnetic particles and erythrocyte membrane by be incubated agent be incubated form.
Further to improve, the incubation agent is Macrogol 4000, and it is 1 that the coupling agent, which is selected from mass ratio,:1-1.2 EDC With NHS mixture.Wherein EDC is 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides, and NHS is N- hydroxy ambers Amber acid imide, bacterial magnetic particles load erythrocyte membrane can be significantly improved as coupling agent by using EDC and NHS mixture Amount.
It is further to improve, in order to further improve the activation effect of bacterial magnetic particles, it is also added into above-mentioned coupling agent Mass ratio is 1:2.5 agar and the mixture of dead plaster.
Further to improve, the incubation agent is that ratio of weight and number is 2:5.5-7.2：2-3 chitosan, sodium alginate and The mixture of Macrogol 4000.The mixture of 3 kinds of materials of selection is remarkably improved bacterial magnetic particles to red thin as agent is incubated The load effect of after birth.
Further to improve, the blood group antigens on erythrocyte membrane keep epitope activity, and blood group antigens include but unlimited Antigen in ABO, Rh, Kell, Duffy, Jk, MNSs, Di or Le.
Bacterial magnetic particles erythrocyte membrane composite particles provided by the invention are used for erythrocyte membrane blood group in clinical examination field The long-term preservation (particularly rare blood group antigen preserves) of antigen, and it is used for blood group as the substitute of fresh reagent panel erythrocyte Application in antibody test.
Another aspect of the present invention also provides a kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles, the preparation side Method comprises the following steps：
1) activate：Add coupling agent to activate bacterial magnetic particles surface, the bacterial magnetic particles after activation are made；
2) it is incubated：Bacterial magnetic particles after activation are mixed with erythrocyte membrane, adds and is incubated agent, incubation at room temperature；
3) magnet adsorption and wash, collect precipitation, produce bacterial magnetic particles erythrocyte membrane composite particles；
Preferably, bacterial magnetic particles first pass through processing step and activated again；
Preferably, the processing step specific method of the bacterial magnetic particles is as follows：
(1) bacterial magnetic particles are taken, adds under inorganic agent stirring condition and reacts, wash product to neutrality after the completion of reaction； The inorganic agent is that volume ratio is 40-60：1-3：The mixed liquor of 0.3-1 absolute ethyl alcohol, ammoniacal liquor and tetraethyl orthosilicate；
(2) bacterial magnetic particles obtained after step (1) is reacted are made into the solution that concentration is 0.1%~0.3%, and regulation is molten Liquid pH value is added in the 3- aminopropyl triethoxysilane ethanol solutions that concentration is 5-15% into above-mentioned solution to 3.0-5.0, React under stirring condition, wash product to neutrality after the completion of reaction, treated bacterial magnetic particles are made；
Preferably, the activation step specific method is as follows：
(a) treated bacterial magnetic particles are taken, add coupling agent composition, stirring reaction filters, and the coupling agent is Mass ratio is 1:1-1.2 EDC and NHS mixture；
Preferably, the condition of incubation is：140-160rpm vortex oscillations, room temperature, reaction is overnight；
Preferably, the incubation agent is Macrogol 4000；
Preferably, the incubation agent ratio of weight and number is 2:5.5-7.2：2-3 chitosan, sodium alginate and polyethylene glycol 4000 mixture.
Another aspect of the present invention additionally provides a kind of preparation method of bacterial magnetic particles, and it comprises the following steps：
A. magnetotactic bacteria MSR-1 bacterial strains are enlarged culture；
B. centrifuge and collect magnetotactic bacteria MSR-1 bacterial strains, suspended with Tris-HCl buffer solutions, suspension is made；
C. the cell in ultrasonic disruption suspension；
D. the cell after magnet adsorption is broken, stands overnight, supernatant discarding, is arrived with phosphate buffer again Suspension adsorption Precipitation, ultrasonic wave cleaning, magnet adsorbs, abandons supernatant, the precipitation being adsorbed onto is suspended with phosphate buffer again, washing 3 It is secondary, produce bacterial magnetic particles；
Preferably,
Centrifugal condition is：Rotating speed 8000-10000rpm, centrifugation time 5-10min；
Ultrasonic disruption condition is：Ultrasonic power 250-350w, ultrasonic time 2-7s, interval time 2-7s, ultrasonic number 80-100 times, it is repeated 3 times；
Ultrasonic wave cleaning condition is：Ultrasonic power 70-90w, ultrasonic time 2-7s, interval time 2-7s, ultrasonic number 40- 60 times.
Another aspect of the present invention additionally provides the preparation method of erythrocyte membrane, and this method comprises the following steps：
A) fresh red blood cell is taken, is washed to supernatant and clarified with NaCl solution, obtains packed red cells；
B) isometric NaCl solution is added into packed red cells, room temperature acts on after mixing, centrifuges and collects precipitation；
C) isometric NaCl solution is added in the precipitation collected to step b), room temperature acts on after mixing, centrifuges and collects Precipitation；
D) isometric distilled water is added in the precipitation received to step c), room temperature acts on after mixing, centrifugation, collects precipitation simultaneously Suspended with PBS, produce erythrocyte membrane.
Another aspect of the present invention additionally provides magnetotactic bacteria MSR-1 bacterial strains and expands cultural method, and it comprises the following steps：
A. the first culture medium, 105-110 DEG C of sterilizing 10min, after standing 1h, by magnetotactic bacteria MSR- are added in culture tank 1 inoculation carries out first time culture into culture tank, obtains the first nutrient solution；The condition of culture is for the first time：Initial ph value For 5.5-6, cultivation temperature is 28-30 DEG C, and incubation time is 3-5 days, and shaking speed is 250-300 revs/min, in incubation Oxygen is passed through, the mode for being passed through oxygen is：20min is spaced, is passed through 50min oxygen；
B. the first nutrient solution is added in the culture tank containing the second culture medium, anaerobic state carries out second and cultivated；The The condition of second incubation is：Initial ph value is 6.5-7.0, and cultivation temperature is 20-25 DEG C, and incubation time is 2-3 days, shaking speed For 100-200 revs/min.
Further to improve, magnetotactic bacteria expands in incubation, and the first culture medium is 5-10 parts by parts by weight Glutamine, 1-3 parts maltitol, 0.5-1 parts calcium pantothenate, 0.5-1 parts casein sodium, 0.2-0.5 parts potassium dihydrogen phosphate, 0.5-1 part laurate methyl amimoacetic acids and 1-2 parts sodium chloride composition；Second culture medium is 2-3 parts peptone, 2-5 parts by parts by weight Biotin, 1-2 parts glycine, 0.5-1 parts sodium tartrate, 1-2 parts ammonium nitrate, 1-2 parts phosphatidyl serine and 0.2-0.5 parts month The composition of cinnamic alcohol sodium sulfovinate.
Ratio of weight and number is added during the incubation that another aspect of the present invention provides as 2:5.5-7.2 chitosan and sea The mixture of mosanom.
The beneficial effects of the present invention are bacterial magnetic particles compared with exposed magnetic material, be easily dispersed, be uniform in size, Surface area is big, and has biomembrane coating outside, is not easy to assemble between particle, has good dispersiveness, superparamagnetism and biology Compatibility.Bacterial magnetic particles are used for the long-term preservation of blood group antigen, a kind of quick, easy blood can be provided for clinic It is clear to learn detection test method.
Embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation Example is only the part of the embodiment of the present invention, rather than whole embodiments.It is common based on the embodiment in the present invention, this area All other embodiment that technical staff is obtained under the premise of creative work is not made, belong to the model that the present invention protects Enclose.
A kind of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 1
The particle is that bacterial magnetic particles after being activated by surface group by coupling agent and erythrocyte membrane are incubated by being incubated agent Educate and form；
The preparation method of the particle is：
1) activate：It is 1 to add mass ratio:1 EDC and NHS mixture activate to bacterial magnetic particles surface, are made Bacterial magnetic particles after activation；
2) it is incubated：Bacterial magnetic particles after activation are mixed with erythrocyte membrane, add Macrogol 4000, incubation at room temperature；
3) magnet adsorption and wash, collect precipitation, produce bacterial magnetic particles erythrocyte membrane composite particles.
A kind of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 2
The particle is that bacterial magnetic particles after being activated by surface group by coupling agent and erythrocyte membrane are incubated by being incubated agent Educate and form；
The preparation method of the particle is：
1) activate：It is 1 to add mass ratio:1.2 EDC and NHS mixture activate to bacterial magnetic particles surface, system Bacterial magnetic particles after must activating；
2) it is incubated：Bacterial magnetic particles after activation are mixed with erythrocyte membrane, it is 2 to add ratio of weight and number:5.5：2 shell The mixture of glycan, sodium alginate and Macrogol 4000, incubation at room temperature；
3) magnet adsorption and wash, collect precipitation, produce bacterial magnetic particles erythrocyte membrane composite particles.
A kind of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 3
The particle is that bacterial magnetic particles after being activated by surface group by coupling agent and erythrocyte membrane are incubated by being incubated agent Educate and form；
The preparation method of the particle is：
(1) bacterial magnetic particles are taken, adds under inorganic agent stirring condition and reacts, wash product to neutrality after the completion of reaction； The inorganic agent is that volume ratio is 40：1：The mixed liquor of 0.3 absolute ethyl alcohol, ammoniacal liquor and tetraethyl orthosilicate；
(2) bacterial magnetic particles obtained after step (1) is reacted are made into the solution that concentration is 0.1%, adjust solution ph To 3.0, added into above-mentioned solution in the 3- aminopropyl triethoxysilane ethanol solutions that concentration is 15%, it is anti-under stirring condition Should, product is washed to neutrality after the completion of reaction, treated bacterial magnetic particles are made；
(3) treated bacterial magnetic particles are taken, it is 1 to add mass ratio:1 EDC and NHS mixtures, stirring reaction, mistake Filter, the bacterial magnetic particles after activation are made；
4) bacterial magnetic particles after activation are mixed with erythrocyte membrane, adds Macrogol 4000, incubation at room temperature；
5) magnet adsorption and wash, collect precipitation, produce bacterial magnetic particles erythrocyte membrane composite particles.
A kind of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 4
The particle is that bacterial magnetic particles after being activated by surface group by coupling agent and erythrocyte membrane are incubated by being incubated agent Educate and form；
The preparation method of the particle is：
(1) bacterial magnetic particles are taken, adds under inorganic agent stirring condition and reacts, wash product to neutrality after the completion of reaction； The inorganic agent is that volume ratio is 40：1：The mixed liquor of 0.3 absolute ethyl alcohol, ammoniacal liquor and tetraethyl orthosilicate；
(2) bacterial magnetic particles obtained after step (1) is reacted are made into the solution that concentration is 0.1%, adjust solution ph To 3.0, added into above-mentioned solution in the 3- aminopropyl triethoxysilane ethanol solutions that concentration is 15%, it is anti-under stirring condition Should, product is washed to neutrality after the completion of reaction, treated bacterial magnetic particles are made；
(3) treated bacterial magnetic particles are taken, it is 1 to add mass ratio:1 EDC and NHS mixtures, stirring reaction, mistake Filter, the bacterial magnetic particles after activation are made；
4) bacterial magnetic particles after activation are mixed with erythrocyte membrane, it is 2 to add ratio of weight and number:7.2：3 chitosan, The mixture of sodium alginate and Macrogol 4000, incubation at room temperature；
5) magnet adsorption and wash, collect precipitation, produce bacterial magnetic particles erythrocyte membrane composite particles.
A kind of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 5
The particle is that bacterial magnetic particles after being activated by surface group by coupling agent and erythrocyte membrane are incubated by being incubated agent Educate and form；
The preparation method of the particle is：
(1) bacterial magnetic particles are taken, 0.1%~0.3% (w/v) concentration is configured to distilled water, is ultrasonically treated 10min；
(2) the above-mentioned bacterial magnetic particles of 30mL are taken, then add 50mL absolute ethyl alcohols, 2mL ammoniacal liquor and the positive silicic acid second of 500 μ L Ester, 5h is reacted under 200rpm stirring conditions, product is washed with deionized to neutrality after the completion of reaction；
(3) bacterial magnetic particles of 0.1%~0.3% (w/v) concentration are prepared with absolute ethyl alcohol, pH value of solution is adjusted with glacial acetic acid Value takes the above-mentioned solution of 20mL to add into 30mL 10%3- aminopropyl triethoxysilane ethanol solutions, in 200rpm to 4.0 3h is reacted under stirring condition, product is washed with deionized to neutrality after the completion of reaction, treated bacterial magnetic is made Grain；
(4) treated bacterial magnetic particles are washed with acetic acid-sodium acetate buffer solution (pH 6.0), and is configured to 1% The suspension of~5% (w/v) concentration；
(5) the above-mentioned bacterial magnetic particles suspensions of 2mL are taken, add EDC solution and 100 μ L that 100 μ L concentration are 10% (w/v) 10% (w/v) NHS solution, room temperature 150rpm vortex oscillations reaction 3h, is then washed with deionized water excessive EDC and NHS, And finally it is suspended into 2mL；
(6) the bacterial magnetic particles suspension after activation is mixed with erythrocyte membrane；
(7) 20% isometric (W/V) Macrogol 4000 is added, room temperature effect 30min, is gently shaken；
(8) magnet adsorption, supernatant is abandoned, precipitation is dissolved in phosphate buffer and washed 3 times, it is red that bacterial magnetic particles are made Cell membrane composite particles.
A kind of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 6
The particle is that bacterial magnetic particles after being activated by surface group by coupling agent and erythrocyte membrane are incubated by being incubated agent Educate and form；
The preparation method of the particle as different from Example 5, adds 20% isometric (W/V) in the step (7) Ratio of weight and number is 2:5.5-7.2：The mixture of 2-3 chitosan, sodium alginate and Macrogol 4000.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 7
This method comprises the following steps：
1) activate：It is 1 to add mass ratio:1.1 EDC and NHS mixture activate to bacterial magnetic particles surface；
2) it is incubated：Bacterial magnetic particles after activation are mixed with erythrocyte membrane, add Macrogol 4000, incubation at room temperature；
3) magnet adsorption and wash, collect precipitation, produce bacterial magnetic particles erythrocyte membrane composite particles.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 8
As different from Example 7, the ratio of weight and number added in step 2) is 2 to this method preparation method:6：2.5 shell The mixture of glycan, sodium alginate and Macrogol 4000, incubation at room temperature.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 9
This method comprises the following steps：(1) bacterial magnetic particles are taken, adds under inorganic agent stirring condition and reacts, reaction is completed Product is washed to neutrality afterwards；The inorganic agent is that volume ratio is 40-60：1-3：0.3-1 absolute ethyl alcohol, ammoniacal liquor and positive silicic acid The mixed liquor of ethyl ester；
(2) bacterial magnetic particles obtained after step (1) is reacted are made into the solution that concentration is 0.1%~0.3%, and regulation is molten Liquid pH value is added in the 3- aminopropyl triethoxysilane ethanol solutions that concentration is 5-15% into above-mentioned solution to 3.0-5.0, React under stirring condition, wash product to neutrality after the completion of reaction, treated bacterial magnetic particles are made；
(3) treated bacterial magnetic particles are taken, it is 1 to add mass ratio:1.2 EDC and NHS mixture, stirring are anti- Should, filtering, the bacterial magnetic particles after activation are made；
4) bacterial magnetic particles after activation are mixed with erythrocyte membrane, adds Macrogol 4000, incubation at room temperature；
5) magnet adsorption and wash, collect precipitation, produce bacterial magnetic particles erythrocyte membrane composite particles.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 10
The preparation method adds ratio of weight and number as 2 as different from Example 9, in step 4):6.5：3 shell gathers The mixture of sugar, sodium alginate and Macrogol 4000, incubation at room temperature.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 11
This method comprises the following steps：
(1) bacterial magnetic particles are taken, 0.1%~0.3% (w/v) concentration is configured to distilled water, is ultrasonically treated 10min；
(2) the above-mentioned bacterial magnetic particles of 30mL are taken, then add 50mL absolute ethyl alcohols, 2mL ammoniacal liquor and the positive silicic acid second of 500 μ L Ester, 5h is reacted under 200rpm stirring conditions, product is washed with deionized to neutrality after the completion of reaction；
(3) bacterial magnetic particles of 0.1%~0.3% (w/v) concentration are prepared with absolute ethyl alcohol, pH value of solution is adjusted with glacial acetic acid Value takes the above-mentioned solution of 20mL to add into 30mL 10%3- aminopropyl triethoxysilane ethanol solutions, in 200rpm to 4.0 3h is reacted under stirring condition, product is washed with deionized to neutrality after the completion of reaction, treated bacterial magnetic is made Grain；
(4) treated bacterial magnetic particles are washed with acetic acid-sodium acetate buffer solution (pH 6.0), and is configured to 1% The suspension of~5% (w/v) concentration；
(5) the above-mentioned bacterial magnetic particles suspensions of 2mL are taken, add EDC solution and 100 μ L that 100 μ L concentration are 10% (w/v) 10% (w/v) NHS solution, room temperature 150rpm vortex oscillations reaction 3h, is then washed with deionized water excessive EDC and NHS, And 2mL is finally suspended into, the bacterial magnetic particles after activation are made；
(6) the bacterial magnetic particles suspension after activation is mixed with erythrocyte membrane；
(7) 20% isometric (W/V) Macrogol 4000 is added, room temperature effect 30min, is gently shaken；
(8) magnet adsorption, supernatant is abandoned, precipitation is dissolved in phosphate buffer and washed 3 times, it is red that bacterial magnetic particles are made Cell membrane composite particles.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 12
The preparation method adds ratio of weight and number as 2 as different from Example 11, in step 7):6.3:2 shell gathers The mixture of sugar, sodium alginate and Macrogol 4000.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 13
As different from Example 7, the preparation method also includes the preparation method of bacterial magnetic particles to the preparation method, and it is wrapped Include following steps：
1) magnetotactic bacteria MSR-1 bacterial strains are enlarged culture；
2) centrifuge and collect magnetotactic bacteria MSR-1 bacterial strains, suspended with Tris-HCl buffer solutions, obtain suspension；
3) cell in ultrasonic disruption suspension；
4) cell after magnet adsorption is broken, stands overnight, supernatant discarding, is arrived with phosphate buffer again Suspension adsorption Precipitation, ultrasonic wave cleaning, magnet adsorbs, abandons supernatant, the precipitation being adsorbed onto is suspended with phosphate buffer again, washing 3 It is secondary, produce bacterial magnetic particles.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 14
As different from Example 13, the preparation method of bacterial magnetic particles comprises the following steps the preparation method：
(1) magnetotactic bacteria MSR-1 bacterial strains are enlarged culture.
(2) bacterial strain (10000rpm, 5min) is collected by centrifugation, is suspended, must suspended with appropriate Tris-HCl buffer solutions (pH7.4) Liquid；
(3) cell in ultrasonic disruption suspension, ultrasonic disruption condition are：Ultrasonic power 300w, ultrasonic time 5s, Interval time 5s, ultrasonic number 90 times, it is repeated 3 times, it is completely broken to cell；
(4) somatic cells after magnet adsorption is broken, after standing overnight, supernatant discarding, with 10mM phosphate buffer weights The precipitation that new Suspension adsorption arrives, ultrasonic wave cleaning, magnet adsorb again, abandon supernatant, and phosphate buffer suspends, repeated washing 3 Secondary, ultrasonic wave cleaning condition is：Ultrasonic power 80w, ultrasonic time 5s, interval time 5s, ultrasonic number 50 times；
(5) pure bacterial magnetic particles ultrasonic wave in distilled water cleans twice, magnet adsorption, by the bacterial magnetic of absorption Grain distillation aqueous suspension；- 20 DEG C save backup after freeze-drying.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 15
As different from Example 7, this method also includes the preparation method of erythrocyte membrane to the preparation method, and it includes as follows Step：
(1) fresh red blood cell is gathered, is washed with 0.9% (W/V) NaCl solution supreme limpid clear；
(2) supernatant is abandoned, 0.6% isometric (W/V) NaCl solution is added into packed red cells, room temperature acts on after mixing 5 minutes, centrifuge (2000rpm, 5min) and collect precipitation；
(3) supernatant to be abandoned, adds 0.2% isometric (W/V) NaCl solution, room temperature acts on 5 minutes after mixing, (3000rpm, 10min) is centrifuged and is collected precipitation；
(4) supernatant is abandoned, adds isometric distilled water, room temperature acts on 5 minutes after mixing, centrifuges (3000rpm, 10min) And collect and precipitate and suspended with PBS, produce erythrocyte membrane.
A kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles of embodiment 16
As different from Example 13, this method also includes the expansion culture of magnetotactic bacteria MSR-1 bacterial strains to the preparation method, It comprises the following steps：
A. the first culture medium, 107 DEG C of sterilizing 10min, after standing 1h, by magnetotactic bacteria MSR-1 bacterium are added in culture tank Strain is inoculated into culture tank, carries out first time culture, obtains the first nutrient solution；The condition of culture is for the first time：Initial ph value is 5.8, cultivation temperature is 30 DEG C, and incubation time is 5 days, and shaking speed is 300 revs/min, and oxygen is passed through in incubation, is passed through The mode of oxygen is：20min is spaced, is passed through 50min oxygen；
B. the first nutrient solution is added in the culture tank containing the second culture medium, anaerobic state carries out second and cultivated；The The condition of second incubation is：Initial ph value is 6.5, cultivation temperature be 25 DEG C, incubation time be 2 days, shaking speed be 150 turns/ Minute；
Wherein, the first culture medium by parts by weight be 7.5 parts glutamine, 2 parts of maltitols, 0.7 part of calcium pantothenate, 0.7 part of casein sodium, 0.3 part of potassium dihydrogen phosphate, 0.7 part of laurate methyl amimoacetic acid and 1.5 parts of sodium chloride compositions；
Second culture medium is 2.5 parts of peptones, 3 parts of biotins, 1.5 parts of glycine, 0.75 portion of wine by parts by weight Stone acid sodium, 1.5 parts of ammonium nitrate, the composition of 1.5 parts of phosphatidyl serines and 0.3 part of laruyl alcohol sodium sulfovinate.
Reference examples 1
The reference examples are as different from Example 16：
The condition of culture is for the first time：Initial ph value is 5, and cultivation temperature is 32 DEG C, and incubation time is 5 days, shaking speed For 200 revs/min, oxygen is passed through in incubation always；
The condition of culture is for the second time：Initial ph value is 7.2, and cultivation temperature is 30 DEG C, and incubation time is 2 days, and shaking table turns Speed is 250 revs/min；
The glutamine, 0.7 part of calcium pantothenate, 0.7 part of casein sodium, 0.3 that first culture medium is 7.5 parts by parts by weight Part potassium dihydrogen phosphate and 1.5 parts of sodium chloride compositions；
Second culture medium is 2.5 parts of peptones, 1.5 parts of glycine, 0.75 part of sodium tartrate, 1.5 parts of nitre by parts by weight The composition of sour ammonium and 0.3 part of laruyl alcohol sodium sulfovinate.
Reference examples 2
The reference examples are as different from Example 16：
The condition of culture is for the first time：Initial ph value is 6.2, and cultivation temperature is 25 DEG C, and incubation time is 5 days, and shaking table turns Speed is 350 revs/min, and oxygen is not passed through in incubation；The condition of culture is for the second time：Initial ph value is 6, and cultivation temperature is 15 DEG C, incubation time is 2 days, and shaking speed is 100 revs/min；
First culture medium is 7.5 parts of glutamine, 2 parts of maltitols, 0.7 part of calcium pantothenate, 0.7 portion of junket by parts by weight Protein acid sodium, 0.3 part of potassium dihydrogen phosphate, 2 parts of agar, 0.7 part of laurate methyl amimoacetic acid and 1.5 parts of sodium chloride compositions；
Second culture medium is 2.5 parts of peptones, 3 parts of biotins, 1.5 parts of glycine, 0.75 part of tartaric acid by parts by weight Sodium, 1.5 parts of ammonium nitrate, 2 parts of agar, 2 parts of glucose, the group of 1.5 parts of phosphatidyl serines and 0.3 part of laruyl alcohol sodium sulfovinate Into.
The erythrocyte blood type antibody test of test example 1
1. red blood cell reverse type detects
It is thin by above-mentioned embodiment preparation A types with the red blood cells of type A of known blood group, Type B red blood cell, O-shaped red blood cell Bacterium magnetic particle erythrocyte membrane composite particles, Type B bacterial magnetic particles erythrocyte membrane composite particles, O-shaped bacterial magnetic particles erythrocyte membrane Composite particles, ABO blood group system reverse type, i.e. blood plasma moderate resistance A, anti-B detection are carried out to substitute fresh red blood cell.
Detecting step is as follows：
(1) clean tube three is taken, marks A, B, 0 respectively；
(2) sample to be checked (serum or blood plasma) 50ul is respectively added；
(3) respective tube be separately added into preparation A type bacterial magnetic particles erythrocyte membranes composite particles, Type B bacterial magnetic particles it is red Cell membrane composite particles, O-shaped bacterial magnetic particles erythrocyte membrane composite particles 50ul；
(4) mix, react at room temperature 1min；
(5) magnet adsorption, vibration test tube and result of determination, if aggegation occurs for bacterial magnetic particles erythrocyte membrane composite particles, It is then positive reaction；Conversely, if aggegation does not occur for bacterial magnetic particles erythrocyte membrane composite particles, for negative reaction.React lattice Office's criterion is as follows：
2. red blood cell irregular antibody detects
Pressed with the O-shaped red blood cell of more person-portions of the clinical significant red cell antigens in covering China of known blood group above-mentioned specific real The mode of applying is prepared into bacterial magnetic particles erythrocyte membrane composite particles, and red blood cell irregular antibody inspection is carried out to substitute fresh red blood cell Survey.
Detecting step is as follows：
(1) clean tube is taken, carries out mark；
(2) sample (serum or blood plasma) 50ul to be checked is added；
(3) above-mentioned bacterial magnetic particles erythrocyte membrane composite particles 50ul is added；
(4) mix, react at room temperature 1min；
(5) magnet adsorption, vibration test tube and result of determination, if aggegation occurs for bacterial magnetic particles erythrocyte membrane composite particles (positive reaction), show to contain the antibody corresponding with the particulate antigen in sample to be checked, if conversely, bacterial magnetic particles red blood cell Aggegation (negative reaction) does not occur for film composite particles, shows to be free of the antibody corresponding with the particulate antigen in sample to be checked.
The bacterial magnetic particles of test example 2 load erythrocyte membrane effect test
Different coupling agents is selected, remaining method prepares compound of bacterial magnetic particles antigen according to the method for embodiment 9 Grain, bacterial magnetic particles are shown in Table 1 to the load effect of erythrocyte membrane；
Influence of the different coupling agents of table 1 to erythrocyte membrane load effect
Different incubation agent is selected, remaining method prepares compound of bacterial magnetic particles antigen according to the method for embodiment 10 Grain, bacterial magnetic particles are shown in Table 2 to the load effect of erythrocyte membrane；
The different influences for being incubated agent to the load effect of erythrocyte membrane of table 2
As can be seen from Table 1, it is coupled by coupling agent different in selection 4, the load capacity of erythrocyte membrane can reach 789 μ g/mg, mixed, or be coupled using the coupling agent outside the present invention, bacterial magnetic when using 2 or 3 kind of coupling agent The amount of the erythrocyte membrane of particulate load significantly reduces；From Table 2, it can be seen that chitosan, marine alga when selection proper ratio When sour sodium and Macrogol 4000 mixing is incubated, load capacity of the bacterial magnetic particles to erythrocyte membrane can be improved.
Different treatment conditions are selected, it is 1 that coupling agent, which is selected from mass ratio,:1：1:2.5 EDC, NHS, agar and anhydrous sulphur The mixture of sour calcium, it is 2 to be incubated agent and be selected from mass ratio:5.5：The mixing of 2 chitosan, sodium alginate and Macrogol 4000 Thing, remaining the step of according to the method for embodiment 14 prepare bacterial magnetic particles antigen composite particles, bacterial magnetic particles are to red blood cell The load effect of film is shown in Table 3；
Influence of the different disposal step of table 3 to erythrocyte membrane load effect
As can be known from Table 3, the processing step of bacterial magnetic particles has to bacterial magnetic particles to the load effect of erythrocyte membrane Very big influence.
The magnetotactic bacteria MSR-1 bacterial strains of test example 3 expand culture situation
1) it is grouped
Test 1 group：Preparation method described in the embodiment of the present invention 16；
Compare 1 group：The preparation method of reference examples 1；
Compare 2 groups：The preparation method of reference examples 2；
Compare 3 groups：A kind of amphimicrobian magnetotactic bacteria and its separation, culture-dependent method and bacterium disclosed in CN1952112 Cultural method disclosed in the extraction of magnetic particle, purification process；
2) cell is counted using trypan blue classics decoration method, cell is 1 × 10 during inoculation4Individual/ml, is counted respectively Quantity after primary cell and expansion culture, the results are shown in Table 4.
The each group magnetotactic bacteria MSR-1 bacterial strain cultured and amplified in vitro result of the tests of table 4
Magnetotactic bacteria cultural method provided by the invention can effective in vitro culture magnetotactic bacteria it can be seen from table MSR-1 bacterial strains.
Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair The equivalent structure or equivalent flow conversion that bright description is made, or directly or indirectly it is used in other related technology necks Domain, it is included within the scope of the present invention.

Claims (2)

1. a kind of preparation method of bacterial magnetic particles erythrocyte membrane composite particles, it is characterised in that methods described includes following step Suddenly：
The preparation of bacterial magnetic particles：
A. magnetotactic bacteria MSR-1 bacterial strains are enlarged culture；The magnetotactic bacteria MSR-1 bacterial strains, which expand culture, includes following step Suddenly：
A) the first culture medium, 105-110 DEG C of sterilizing 10min, after standing 1h, by magnetotactic bacteria MSR-1 bacterium are added in culture tank Strain is inoculated into culture tank, carries out first time culture, obtains the first nutrient solution；The condition of culture is for the first time：Initial ph value is 5.5-6, cultivation temperature are 28-30 DEG C, and incubation time is 3-5 days, and shaking speed is 250-300 revs/min, are led in incubation Enter oxygen, the mode for being passed through oxygen is：20min is spaced, is passed through 50min oxygen；
B) the first nutrient solution is added in the culture tank containing the second culture medium, anaerobic state carries out second and cultivated；Second The condition of culture is：Initial ph value is 6.5-7.0, and cultivation temperature is 20-25 DEG C, and incubation time is 2-3 days, and shaking speed is 100-200 revs/min；Glutamine, 1-3 part maltitol, 0.5-1 of first culture medium by parts by weight for 5-10 parts Part calcium pantothenate, 0.5-1 parts casein sodium, 0.2-0.5 parts potassium dihydrogen phosphate, 0.5-1 part laurate methyl amimoacetic acids and 1-2 part chlorinations Sodium forms, and second culture medium is 2-3 parts peptone, 2-5 parts biotin, 1-2 parts glycine, 0.5-1 parts by parts by weight Sodium tartrate, 1-2 parts ammonium nitrate, 1-2 parts phosphatidyl serine and 0.2-0.5 parts laruyl alcohol sodium sulfovinate composition；
B. centrifuge and collect magnetotactic bacteria MSR-1 bacterial strains, suspended with Tris-HCl buffer solutions, suspension, rotating speed 8000- is made 10000rpm, centrifugation time 5-10min；
C. the cell in ultrasonic disruption suspension, ultrasonic power 250-350w, ultrasonic time 2-7s, interval time 2-7s, surpass Sound number 80-100 times, is repeated 3 times；
D. the cell after magnet adsorption is broken, stands overnight, supernatant discarding, and it is heavy to be arrived with phosphate buffer again Suspension adsorption Form sediment, ultrasonic wave cleaning, magnet adsorbs, abandons supernatant, the precipitation being adsorbed onto is suspended with phosphate buffer again, washing 3 times, i.e., Bacterial magnetic particles are obtained, ultrasonic wave cleaning condition is：Ultrasonic power 70-90w, ultrasonic time 2-7s, interval time 2-7s, ultrasound time Number 40-60 times；
1) activate：
Bacterial magnetic particles processing：
(1) bacterial magnetic particles are taken, adds under inorganic agent stirring condition and reacts, wash product to neutrality after the completion of reaction；It is described Inorganic agent is that volume ratio is 40-60：1-3：The mixed liquor of 0.3-1 absolute ethyl alcohol, ammoniacal liquor and tetraethyl orthosilicate；
(2) bacterial magnetic particles obtained after step (1) is reacted are made into the solution that concentration is 0.1%~0.3%, adjust pH value of solution Value adds in the 3- aminopropyl triethoxysilane ethanol solutions that concentration is 5-15%, stirring to 3.0-5.0 into above-mentioned solution Under the conditions of react, product is washed to neutrality after the completion of reaction, be made treated bacterial magnetic particles；
(3) treated bacterial magnetic particles are taken, add coupling agent composition, stirring reaction, filtering, the coupling agent is quality Than for 1:1-1.2 EDC and NHS mixture, the bacterial magnetic particles after activation are made；
2) it is incubated：Bacterial magnetic particles after activation are mixed with erythrocyte membrane, adds and is incubated agent, it is 2 to be incubated agent ratio of weight and number: 5.5-7.2：The mixture of 2-3 chitosan, sodium alginate and Macrogol 4000,140-160rpm vortex oscillations, room temperature are incubated Educate, reaction is overnight；
3) magnet adsorption and wash, collect precipitation, produce bacterial magnetic particles erythrocyte membrane composite particles.
2. the preparation method of bacterial magnetic particles erythrocyte membrane composite particles as claimed in claim 1, it is characterised in that described red The preparation method of cell membrane is：
A) fresh red blood cell is taken, is washed to supernatant and clarified with NaCl solution, obtains packed red cells；
B) isometric NaCl solution is added into packed red cells, room temperature acts on after mixing, centrifuges and collects precipitation；
C) isometric NaCl solution is added in the precipitation collected to step b), room temperature acts on after mixing, centrifuges and collects precipitation；
D) isometric distilled water is added in the precipitation received to step c), room temperature acts on after mixing, centrifugation, collects precipitation and is used in combination PBS suspends, and produces erythrocyte membrane.
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